Chemotherapy Preconditioning Fails to Boost Inhaled IL-15 Immunotherapy in Dogs with Lung Metastases

In a finding that tempers one of the more hopeful threads in veterinary immunotherapy, researchers at the University of California, Davis have shown that priming the immune system with chemotherapy before inhaled interleukin-15 treatment does not improve outcomes in dogs with advanced lung metastases. The study, published in Veterinary Oncology, tested whether a short course of doxorubicin could strip away competing immune cells and give the cytokine therapy a clearer field in which to work. The answer, based on ten carefully enrolled canine patients, was a cautious but clear no.

The rationale behind the trial was grounded in a genuine biological puzzle. In an earlier phase 1 study, inhaled recombinant human interleukin-15, or rhIL-15, proved safe in dogs with metastatic osteosarcoma and melanoma and produced durable responses in a subset of animals. Intriguingly, dogs that entered the trial with lower absolute lymphocyte counts were statistically more likely to benefit. The Davis team hypothesized that reducing lymphocytes before treatment might remove so-called cytokine sinks, circulating immune cells that soak up interleukin-15 before it can reach the natural killer cells and CD8-positive memory T cells the drug is meant to energize.

Lymphodepletion is a well-established strategy in human adoptive cell therapy, where chemotherapy is routinely used before infusing engineered T cells to improve their expansion and persistence. Doxorubicin, a mainstay chemotherapy agent in veterinary medicine, had previously been shown to reduce circulating T lymphocytes in tumor-bearing dogs. The researchers therefore designed a trial in which dogs received a standard dose of doxorubicin, 30 milligrams per square meter for larger animals or 1 milligram per kilogram for smaller ones, followed one week later by two weeks of inhaled rhIL-15 at 50 micrograms twice daily, delivered by nebulizer at home by trained owners.

The study population consisted of ten client-owned dogs with histologically or cytologically confirmed melanoma or bone tumors, all carrying at least one lung lesion of a centimeter or larger on thoracic radiographs. Five dogs had osteosarcoma, four had melanoma, and one had a multilobular tumor of bone. Eligibility criteria were strict: dogs needed adequate organ function, no prior rhIL-15 exposure, no recent chemotherapy or immunotherapy, and screening echocardiograms to rule out cardiac disease that could be worsened by doxorubicin. The trial was approved by the UC Davis institutional review boards, and all owners provided informed consent.

When the results came in, the numbers told a sobering story. Two dogs progressed before the Day 35 response assessment, leaving eight evaluable patients. One dog achieved a complete response that has now lasted more than two years, two had stable disease, and five progressed. That translates to an overall response rate of 10 percent and a clinical benefit rate of 30 percent, figures essentially indistinguishable from the phase 1 trial of inhaled rhIL-15 alone, which reported a response rate of 11 percent and a clinical benefit rate of 39 percent. The trial had been powered to detect a jump in response rate from 11 to 50 percent, so the null result is unlikely to be a statistical fluke of modest effect size.

The hematological data confirmed the team did achieve the biological precondition they set out to create. Seven days after doxorubicin, mean absolute lymphocyte counts fell from 1,452 to 1,023 cells per microliter, a statistically significant drop that brought the dogs to lymphocyte levels comparable to those seen in responding patients from the earlier trial. White blood cells, monocytes, and neutrophils also declined significantly, and one dog developed grade 4 neutropenia with fever that resolved within two days of hospitalization and supportive care. Crucially, adding doxorubicin did not increase the toxicity of the inhaled cytokine therapy itself, and no allergic reactions or pulmonary adverse events were attributed to rhIL-15.

Transcriptomic analysis added an unexpected layer of insight. Using the NanoString nCounter canine immuno-oncology panel covering 780 genes, the researchers profiled peripheral blood mononuclear cells collected before doxorubicin, after doxorubicin, and after two weeks of inhaled rhIL-15. While overall lymphocyte counts fell, the relative proportions of T cells, CD8-positive T cells, exhausted CD8-positive T cells, natural killer cells, and Th1 cells did not change significantly, indicating that doxorubicin depleted lymphocytes indiscriminately rather than selectively removing suppressive populations. The one clear casualty was the B cell compartment: B cell scores and B cell function genes, including CD19, CD79B, MS4A1, and immunoglobulin genes, dropped significantly and had not recovered by Day 21, consistent with the rapid proliferation rate of B cells making them especially vulnerable to chemotherapy.

Perhaps the most tantalizing clue came from the single dog that achieved a complete response. That patient initially appeared to progress at Day 35, with new lung lesions appearing on radiographs, but after receiving palliative radiation to its primary mandibular melanoma, every lung lesion resolved by week 15. The researchers suggest this may represent pseudoprogression, a well-recognized phenomenon in human immunotherapy where apparent tumor growth reflects immune cell infiltration rather than true malignancy, though an abscopal effect triggered by the radiation cannot be excluded. Transcriptomically, this dog trended toward higher T cell, CD8-positive T cell, and cytotoxic cell scores, and started with a lower exhausted CD8 T cell score than non-responders, hinting that baseline immune fitness may predict who benefits from inhaled rhIL-15.

The study carries important limitations that the authors acknowledge candidly. Ten patients is a small cohort, and two dogs did not survive to formal response assessment. The therapy used human rather than canine interleukin-15, which likely provoked neutralizing antibodies, although the two-week treatment window was chosen to minimize that risk. Target lesions smaller than the standard 2-centimeter RECIST threshold were permitted, matching the phase 1 design but introducing radiographic measurement imprecision. Tumor type may also have influenced outcomes, since one of the stable disease patients had a multilobular tumor of bone, an inherently slower-growing cancer.

Even so, the trial delivers a valuable double message. First, it definitively rules out standard-dose doxorubicin preconditioning as a way to enhance inhaled rhIL-15 efficacy, redirecting the field toward other combination strategies such as radiation, which has already shown promise with cytokine therapy in canine sarcoma and melanoma. Second, it confirms that durable, multi-year complete responses are achievable even in dogs with diffuse pulmonary metastatic disease, a population whose prognosis otherwise hovers below 80 days. For the dogs that respond, and for the human osteosarcoma and melanoma patients their disease so closely mirrors, that signal is worth chasing with better-designed combinations.

Subject of Research: Doxorubicin preconditioning and inhaled recombinant human IL-15 immunotherapy in dogs with pulmonary metastasis

Article Title: Evaluating the impact of doxorubicin preconditioning on the efficacy of inhaled recombinant human IL-15 immunotherapy in dogs with pulmonary metastasis

Article References: Luker, M. E., York, D., Al-Nadaf, S., Johnson, E. G., Withers, S. S., Cruz, S. M., Lejeune, A., Skorupski, K. A., Young, J., Cohen-Davidyan, T., Sparger, E. E., Culp, W. T., Murphy, W. J., Kent, M. S., Canter, R. J., & Rebhun, R. B. (2025). Evaluating the impact of doxorubicin preconditioning on the efficacy of inhaled recombinant human IL-15 immunotherapy in dogs with pulmonary metastasis. Veterinary Oncology, 2(1), Article 23. https://doi.org/10.1186/s44356-025-00040-5

Image Credits: AI Generated

DOI: 10.1186/s44356-025-00040-5

Keywords: immunotherapy, interleukin-15, doxorubicin, canine osteosarcoma, melanoma, lung metastasis, lymphodepletion, veterinary oncology, natural killer cells, CD8 T cells, pseudoprogression, transcriptomics